ABSTRACT
Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.
Subject(s)
Alternative Splicing , Apoptosis/genetics , Cardiovascular Diseases/genetics , Gene Expression Regulation , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Cardiovascular Diseases/diagnosis , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/therapy , Humans , Signal TransductionABSTRACT
Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).
Subject(s)
Avian Proteins , Cathepsin D , Food Handling , Meat/analysis , Muscle Proteins , Muscle, Skeletal/enzymology , Struthioniformes/metabolism , Amino Acid Sequence , Animals , Avian Proteins/antagonists & inhibitors , Avian Proteins/chemistry , Avian Proteins/isolation & purification , Avian Proteins/metabolism , Cathepsin D/antagonists & inhibitors , Cathepsin D/chemistry , Cathepsin D/isolation & purification , Cathepsin D/metabolism , Chromatography, Affinity , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Male , Molecular Sequence Data , Molecular Weight , Muscle Proteins/antagonists & inhibitors , Muscle Proteins/chemistry , Muscle Proteins/isolation & purification , Muscle Proteins/metabolism , Pepstatins/metabolism , Pepstatins/pharmacology , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Protein Subunits , Sequence Alignment , Sequence Homology, Amino Acid , TemperatureABSTRACT
A myofibril-bound serine protease (MBSP) was partially purified from ostrich (Struthio camelus) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 degrees C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters (K(m) and V(max) values) were calculated from Lineweaver-Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%).
